Method and device for measuring blood coagulation or lysis by viscosity changes

ABSTRACT

The present invention provides a single-use electronic device and test card for use therein which performs a coagulation or lysis assay of a blood sample. The device includes a housing having an exterior surface and defining an interior area and means for receiving the sample through the housing into the interior space. A non-porous substrate is positioned within the interior space for receiving the sample thereon. A reagent accelerates the coagulation of the sample and is positioned on the substrate and in contact with the sample. Optionally, an electroactive species may also be reacted with the sample. The device also includes means for measuring the viscosity of the sample and generating an electrical signal which correlates to a curve of the coagulation/lysis assay. Processing means positioned within the interior space is connected to the measuring means for receiving and converting the electrical signal into a digital output corresponding to the coagulation/lysis assay using assay calibration information stored therein. Display means visually displays the digital output external to the housing and is connected to the processing means. A method of determining the coagulation or lysis of a sample provides for accelerating the coagulation of the sample by chemically reacting the sample with at least one reagent on the substrate to produce a detectable change in the viscosity of the sample which correlates with the state of coagulation or lysis of the sample and measuring the viscosity of the sample and generating an electrical signal which correlates to a curve of the coagulation/lysis assay.

FIELD OF THE INVENTION

The present invention relates to a device and method for preciselymeasuring the coagulation or lysis of blood at the point-of-care. Moreparticularly, a diagnostic device which is wholly or partly disposableis used to measure the viscosity change through the electricalconductivity of the blood or the diffusion coefficient or electricalconductivity of an electroactive species in the blood to perform acoagulation or lysis assay and display the assay results in real-time.

BACKGROUND OF THE INVENTION

The mechanism by which the body prevents loss of blood from the vascularsystem is known as hemostasis. Blood maintains a state of fluidity innormal circulation, but forms a barrier when trauma or pathologicconditions cause vessel damage. Coagulation tests measure the blood'sability to form a clot or coagulate and are used to manage a patient'santicoagulant therapy and diagnose hemostatic disorders. Lysis testsmeasure the reverse change where one is measuring the lytic activity ofcoagulated blood which is broken down to soluble degradation productsby, for example, an enzyme plasmin.

There are two well-recognized coagulation pathways: the extrinsic orthromboplastin-controlled and the intrinsic or prothrombin/fibrinogen-controlled coagulation pathway. Both the extrinsic andintrinsic pathways result in the production of thrombin, a proteolyticenzyme which catalyzes the conversion of fibrinogen to fibrin. Tworoutine coagulation tests measure the Prothrombin Time (PT) and theActivated Partial Thromboplastin Time (APTT). Both tests measureclotting time to evaluate a patient's baseline hemostatic state or tomonitor the response to anticoagulant therapy as well as the overallfunction and status of the coagulant system.

The PT test is used to assess the extrinsic and common pathway clottingsystems and for monitoring long term anticoagulant therapy. A commonmedication for long term anticoagulant therapy is sodium warfarinisopropanol clathrate, generally known by the brand name COUMADIN®, madeby Dupont Pharmaceuticals of Wilmington, Del. Warfarin and its analogsinduce anticoagulation by effectively blocking biosynthesis of Vitamin Kdependent coagulation factors. Since the PT test measures clotting time,the effective amount of anticoagulant in the blood can be determined.

Another common medication which is in connection with cardiac bypasssurgery, cardiac catheterization, renal dialysis, and in critical caresituations for acute myocardial infarction is Heparin. The APTT test iswidely used test for monitoring Heparin therapy for screeningdeficiencies of clotting factors included in the intrinsic and commoncoagulation system. Heparin exerts its anticoagulation effect by bindingto and forming a complex with a plasma cofactor called antithrombin III.

Many laboratory clotting tests in the prior art are based on thephenomenon of measuring an endpoint which is a change of phase when atest sample changes from a liquid to a coagulated form. This phasechange is due to the conversion of a soluble plasma protein fibrinogento insoluble fibrin by the action of the enzyme thrombin. The clottingendpoint is physically detected by such secondary indicators as color orfluorescence detection by optical means and turbidity measurementsthrough light scattering and magnetic particle oscillation. Theselaboratory instruments are relatively large because of the complextechnology used, expensive, and designed for use by trained personneldue to the complexity of the detection methods. See generally U.S. Pat.No. 5,344,754. Large blood samples are also usually required.

Some prior art devices use porous membrane supports impregnated withlayers of a reagent for enzymatic assays which rely on monitoring theintensity of the reaction product by optical spectroscopy such asreflectance, fluorescence, luminescence or color change. Such reagentimpregnated membranes increase the complexity of the reaction'senvironment due to the absence of a liquid phase which is the idealenvironment for reactions or phase transitions, and could further leadto possible interference with the enzymatic pathway. The accuracy of theresults from membrane based systems are further affected by bloodhematocrit and reaction volumes. The need for larger liquid samples ofblood to achieve complete wetability of the membrane imposes yet anotherconstraint. To overcome some of the problems with membranes, U.S. Pat.No. 5,418,141 teaches the use of expensive, high purity reagents.However, coagulation techniques which use Thrombin substrate chemistriessuffer from a major drawback due to their insensitivity to fibrinogendeficiencies which could yield inaccurate clotting times.

Many studies of blood coagulation have attempted to demonstrate thatmeasuring blood resistance detects clotting time and obtains aquantitative measurement of the rate of clot retraction. The resultswere usually not reproducible and there was "considerable variation andinconsistency in most methods in common use" as disclosed in Rosenthal,R. L., and Tobias C. W.: Measurement of the Electrical Resistance ofHuman Blood; Use in Coagulation Studies, J Lab Clin Med 33, 1110, 1948.Critical emphasis was placed upon the geometric orientation of the cellwithin which a pool of the blood sample was retained and the electrodes.It was also critical to prevent vibration of the cell. It was observedthat as soon as the blood clotted, clot retraction begins by thecontraction of the fibrin network which pulls the large elements orcells together into a dense mass, thus displacing the serum to theperiphery. This process produces increases in resistance measurementsbecause it simultaneously increases the concentration of poorlyconducting cells and decreases the concentration of serum, a goodconductor, between and around the electrodes. Prior to clot formationthere is no significant change in resistance. The clotting time andstart of clot retraction are marked by the first increase in resistance.Thus the clotting time may be determined only with the elimination ofmotion. Subsequent increases in resistance resulted from retraction ofthe clot. The slope of the rising portion of the time-resistance curvewas assumed to correspond to the rate of clot retraction.

As later disclosed in Hirsch FG, et al: The Electrical Conductivity ofBlood I. Relationship to Erythrocyte Concentration, Blood 5: 1017, 1950,"Some workers have attributed these changes to the effects ofcoagulation,³⁰⁻³⁴ but others were unable to confirm these findings.³⁵,36with certain designs of conductivity cells, blood resistance wasobserved to increase due to extrusion of serum during clotretraction,³²,35. Blood conductivity was also found to vary withsedimentation,³⁵,37,38 agitation,³⁷ or stirring.³⁹⁻⁴² " It is furtherreported in Table I on page 1018 that the conductivity was unchangedduring clotting and that only during clot retraction that there was adecrease in conductivity.

As disclosed in U.S. Pat. No. 4,947,678, a device measures viscositychanges in blood to determine blood coagulation. An electricallyconductive sensor heats a blood sample by passing current through thesample. The temperature of the sensor is averaged using its surfacetemperature and the current applied to the sensor. The sampletemperature is also monitored and the difference between it and theaverage sensor temperature is calculated. Changes in the calculatedtemperature difference is used as the indication of viscosity change.

Thus, a need exists in the field of diagnostics for a method and devicefor measurement of blood coagulation or lysis which is sufficientlyinexpensive, timely, efficient, convenient, durable, and reliable foruse in a diagnostic device which permits point-of-care use by untrainedindividuals in locations such as the home, sites of medical emergencies,or locations other than a clinic. Whether the device is disposable orreusable, there is also a need to operate with small blood sample sizes.

SUMMARY OF THE INVENTION

The present invention provides an electrode assembly which providesquantitative measurement of viscosity changes over intervals of time tosignal the coagulation or lysis of a blood sample. The viscosity changeis determined in real time by either the electrical conductivity of theblood or the diffusion coefficient or electrical conductivity of anelectroactive species in the blood sample.

The present invention further provides a single-use electronic devicefor performing a coagulation or lysis assay of a blood sample. Thedevice includes a housing having an exterior surface and defining aninterior area and means for receiving the sample through the housinginto the interior space. A non-porous substrate is positioned within theinterior space for receiving the sample thereon. A reagent acceleratesthe coagulation of the sample and is positioned on the substrate and incontact with the sample. The device also includes means for measuringthe viscosity of the sample and generating an electrical signal whichcorrelates to a curve of the coagulation/lysis assay. Processing meanspositioned within the interior space is connected to the measuring meansfor receiving and converting the electrical signal into a digital outputcorresponding to the coagulation/lysis assay using assay calibrationinformation stored therein. Display means visually displays the digitaloutput external to the housing and is connected to the processing means.

A test card for the above electronic device is provided which generatesa signal having a pre-determined frequency and determines a curve of acoagulation or lysis assay of a blood sample. The test card includes anon-porous substrate defining a surface for receiving the sample. Areagent is initially immobilized on the surface for contact with thesample. A plurality of electrodes are positioned on the substrate andcontact the sample. The electrodes are adapted to receive and pass thesignal into the sample. The electrodes generate an electrical signalcorresponding to the viscosity of the sample which correlates to thecurve of the coagulation/lysis assay.

A method of determining the coagulation or lysis of a sample is alsoprovided by the present invention. The steps of the method include:introducing the sample to a sample receptor site on a substrate;accelerating the coagulation of the sample by chemically reacting thesample with at least one reagent on the substrate to produce adetectable change in the viscosity of the sample which correlates withthe state of coagulation or lysis of the sample; measuring the viscosityof the sample and generating an electrical signal which correlates to acurve of the coagulation/lysis assay; and, processing the electricalsignal into a digital output corresponding to the coagulation/lysisassay using assay calibration information.

The present invention also provides an electroactive species positionedon the substrate and in contact with the sample which enhances thesensitivity of measuring the electrolytic properties of the bloodsample. Determining the electrolytic properties of the sample caninclude measuring the conductivity/resistivity or the diffusioncoefficient of the electroactive species. Preferred electroactivespecies include ferricyanide, ferrocyanide, cadmium chloride, andmethylviologen.

The present invention provides a coagulation measurement device which issufficiently compact and inexpensive for use in a diagnostic device thatis portable and disposable after a single use. The device also providesprecise and accurate measurement of the reaction chemistry of thediagnostic device with results provided in a timely manner for theconvenience of the user.

The advantages, embodiments, variations and the like will be apparent tothose skilled-in-the-art from the present specification taken with theaccompanying drawings and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which comprise a portion of this disclosure:

FIG. 1 is a perspective view of a diagnostic device of the presentinvention;

FIG. 2 is a schematic view of the diagnostic device illustrated in FIG.1;

FIG. 3 is an embodiment of a disposable test card of the presentinvention for insertion into the device illustrated in FIG. 1 with aninitially immobilized reagent and electrodes for measuring the viscositychanges of a blood sample;

FIG. 4 is an embodiment of the test card as illustrated in FIG. 3 with acapillary channel for drawing a blood sample into contact with theelectrodes;

FIG. 5 is an embodiment of the test card of the present invention havingtwo sets of electrodes in distinct testing areas for performing aseparate on-board control test;

FIG. 6 is another embodiment of a test card of the present inventionhaving a working electrode, a counter electrode and a referenceelectrode;

FIG. 7 is a cross-sectional view of a test card of the presentinvention;

FIG. 8 is graph of conductivity in mS/cm vs. Time in seconds whichillustrates a change in conductivity with increasing viscosity as awhole blood sample coagulates;

FIG. 9 is a graph of conductivity in mS/cm vs. time in seconds whichillustrates the change in conductivity during the test;

FIG. 10 compares a normal plasma sample which has been citrated (CNP)with a heparinised normal plasma sample (HNP) and a citrated COUMADINplasma sample (CCP) in a graph of conductivity vs. time; and

FIG. 11 illustrates the clotting profiles of whole blood samplescomparing a Heparinized sample and a citrated sample in a graph ofconductivity vs. time.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is preferably utilized in the disposable,single-use digital electronic instrument and assay devices. However,another preferred embodiment of the present invention uses amultiple-use or reusable device which is compact for hand-held operationor easy portability and is adapted to receive a disposal test cardinserted therein. The present invention provides for the precise andaccurate measurement of electrical or diffusion properties from one ormore sampling areas located on one or more test cards to quantitativelyperform a coagulation or blood lysis assay. The sampling areas can beone or more detection zones exhibiting a detectable change in theelectrical or diffusion properties of the sample corresponding to thestate of coagulation or lysis of the sample.

One embodiment of a reusable diagnostic device 10 of the presentinvention is illustrated in FIGS. 1 and 2. The device 10 includes ahousing 12 having an inlet port 14 for receiving a test card 16 for atleast partial insertion therethrough. The test card 16 is preferablydisposal after performing the desired assay. The term test card 16 asused herein refers to any assay strip, cartridge, or other geometricshape which can support one or more reagents and electrodes as describedherein. The device 10 itself may assume any convenient geometric shapeas long as the electronics and chemistry described herein are costeffectively contained with acceptable performance.

FIG. 2 schematically illustrates a preferred embodiment of a circuit andthe discrete electronics which control the measuring of electrical ordiffusion properties of the sample during the coagulation or lysisassay. Mounted in the interior space of the housing 12 are all of thecomponents, including a power supply 28 required to conduct the assay.Optionally, the device may provide a plug for an AC adaptor. The testcard 16 inserts into the device and is positioned in thermal proximityto a heater 18 which is used to warm the sample on the test card to apre-determined temperature above room temperature. Any conventionalheater 18 of the appropriate size and heating capacity for theanticipated sample size is suitable. One example of a suitable heater 18is manufactured by Minco which makes thermofoil heaters. The thermofoilheaters apply heat precisely and accurately directed to the test card 16in the proximity of the sample without actually needing to touch thesample itself The thermofoil heaters contain thin and flexible heatingelements sandwiched between a flexible insulation such as Kapton C. Theelement of the heater 18 is a flat foil instead of a tubular wire.Preferably, the thermofoil heater is mounted between or below asubstrate such as aluminum or other thermally conductive material forefficient transfer of heat to the test card 16. Preferably, thetemperature is maintained at 37° C. so that the test results can readilycompared to other standardized test results without interpolation.

A temperature sensor 24 is mounted in proximity to the detection orsampling area 20 where the sample is applied or transported to in orderto detect the temperature of the system and provide ambient temperatureinformation for calibration adjustment at temperature extremes. It issuitable to locate the temperature sensor 24 anywhere in or on thedevice. For example, the temperature sensor 24 may be located on thetest card 16.

The power supply 28 has a lead from its negative pole connected to oneside of a electrode pair 22, and a lead from its positive pole beingconnected to an analog to digital converter 30 and display 26. Aprocessor and memory component 32 is connected to the analog to digitalconverter 30 and the display 26. External ports 34 are connected to theanalog to digital converter 30 for receiving assay calibrationinformation, interfacing with a computer, or downloading test results.One end 38 of each of the electrode pair 22 electrically connects to acorresponding pair of contact pads 40 which provide connection to theprocessor 32 and the power source 28. The connection between theelectrodes 22 and the contact pads 40 is made when the test card 16 isinserted into the inlet port 14.

The detection or sampling area 20 is configured to receive the sampledirectly or have the sample transported into the area. The detectionarea 20 includes one or more reagents which are initially immobilized onthe surface of the test card 16 in the area. Upon application of thesample to the detection area 20, one or more of the reagents is wet bythe sample and mixing therewith. The detection area 20 is alsoconfigured to retain the sample in contact with electrodes 22. Thesample can also be treated by additional reagents or filtered before thesample is placed in contact with the electrodes 22.

Optionally, the test card 16 has an electrode pair 36 mounted thereon inproximity to the detection area 20, to detect the presence and movementof sample liquid on the test card 14. Presence of the sample liquidbridging the electrode pair 36 reduces the resistance across theelectrodes, signaling the presence of a conductor (sample liquid)therebetween. When the sample contacts the electrodes 36 the chemicalreactions are well underway and the instrument begins to read thereagent system. The reading may begin immediately when the samplecontacts the electrodes 36 or there may be some time delay of about lessthan 1 second to 10 minutes (preferably from about 30 seconds to 2minutes). There may be single or multiple readings or the readings maycontinue until the reagent system response has stabilized either to anendpoint, maximum or minimum, or to a constant reaction rate.

The processor 32 can be any common or custom integrated circuit withmemory. The processor 32 must have the capacity to either store a set ofpre-programmed calibration information or have the capability to beprogrammed during device manufacturing. In the case of preprogrammedcalibration, selection of appropriate information during manufacture isnecessary and can be done by laser burning of a selection of circuitpathways or any convenient means. In the case of post-manufacturecalibration, a method to load calibration data onto the chip isnecessary, for example external ports 34. External calibration can beaccomplished with external electrical contacts or may be done with anon-contact method using radio waves, magnetic fields, pulse light,laser or the like. The non-contact method of calibration may be morepractical and efficient from a manufacturing viewpoint.

The processor 32 will also control the entire operation of theinstrument including, but not limited to, turning the instrument on inresponse to insertion of a test card 16, providing electrical power ortime signals; timing with an on-board clock, recording, and processingthe instrument zero function; controlling any time delays or timed stepsduring reading; determining when the assay has stabilized; receiving andprocessing information from the temperature sensor; and receiving inputfrom measuring the electrical properties of the sample and converting itto output, based on calibration information, to the display. Theprocessor will also determine if the coagulation or lysis reaction hasoccurred within the specified time, to a specified endpoint range orwithin a specified reaction rate range to control for inactive reagents.Any other electronic control checks can also be included. The processor32 includes codes which identify the manufacturing lot numbers of thedevice components. The processor 32 contains a program which includes,but is not limited to, interpreting the current off the electrodes,relating the signal strength ratio to the reference strength, providingassay results, identifying potential errors, and performing otherquality control checks.

Using these measurements with information stored in the processor 32accurate results upon completion of each assay. Examples of theinformation stored in the microprocessor includes, but is not limitedto, algorithms or calibration curves for the analytes selected foranalysis and other assay calibration information; reactionstabilization, endpoint, or rate information; and manufacturing lotinformation on each of the chemical reagents, detectors, LEDs, assaystrips, and other components used in the device.

The power supply 28 can be any convenient device including, but notlimited to, a battery or a solar cell. The shelf life of the finalproduct will be about 6 months to about 24 months at room temperature.The power supply must have stability consistent with this shelf life.

The display 26 preferably is a liquid crystal device LCD or anyconventional, inexpensive display device. The number size in the displayshould be sufficiently large to allow most people to read the assayvalue, even if they have poor vision. The display height can be about2.0 cm. The number of digits in the display can be anywhere from 1 toabout 10 digits, however, a 3 to 5 digit display is usually sufficient.In addition to showing the assay result, the display may show messagesrelating to the assay result or processing or give error messages

The converter 30 can include a multiplexer to integrate the signal fromthe electrodes and provide the digital signal to the processor 32. Theprocessor can be used to count the time required for the integral toreach a fixed voltage comparator threshold. The time is proportional tothe average signal over the sampling period.

In another preferred embodiment of the present invention, the entiredevice 10 can be disposable by having the test card 16 assembled as partof the device and sealed with all of the other components within theinterior space of the housing during manufacturing. To introduce theblood sample onto the detection area 20 of the test card 16, a receptorport 44 seen in FIG. 1 extends from the surface of the housing 12 to itsinterior for receiving a sample. Once the sample is introduced throughthe receptor port 44, the sample is chemically reacted with at least onereagent 42 to produce a reaction product mixture corresponding to thestate of coagulation or lysis of the sample. A portion of the reactionproduct mixture produces a detectable change in the electrical ordiffusion properties of the sample which correlates with the coagulationor lysis state of the sample. Although the device 10 can be activatedautomatically by the insertion of the test card 16, a manual test button46 can be optionally provided on the housing 12 to be externallyaccessible.

The device 10 can be of any convenient size with the optimal dimensionsdetermined by several factors including convenience of use to theconsumer. Preferably, the device 10 has a volume range of about 5 cm³ toabout 500 cm³.

In the operation of one of the preferred embodiments of the presentinvention, the viscosity changes of a sample is determined by reactingthe sample, within the housing of at least partially disposable device,with a reagent corresponding to assay for the sample to yield adetectable change in electrical or diffusion properties which correlateswith the coagulation or lysis assay for the sample. Subsequently, theviscosity change is calibrated using the assay calibration informationpreviously described and transformed to a numerical output. The assaycalibration information is uniquely characteristic to the specificreagent in the housing and to the detectable change in electrical ordiffusion properties for each sample.

The term specific reagent refers to the reagent contained in theindividual device housing. For the single-use device which is entirelydisposable, the chemistry (i.e. manufacturing lot number, etc.) of thespecific reagent is known when the housing, interior components, andreagent are manufactured. As a result, the present invention can useassay calibration information that is unique to the specific reagent.Similarly, the assay calibration information can include specific,individual information on each component used in manufacturing theindividual assay device. Preferably, the device is manufactured with theassay calibration information stored in the processor within the housingand all of the components sealed in the housing. For the single-use testcard and reusable device, this information can be encoded on to the testcard and read by the device upon insertion.

The assay calibration information can be used to determine the accuracyof the assay by measuring an electrical signal produced in response tothe detectable change measuring viscosity through electrical ordiffusion properties with a pre-determined range for the electricalsignal. The detectable change can also be calibrated to a referencestandard contained in or calculated using the assay calibrationinformation. The assay results can also be adjusted to the ambienttemperature of the device housing using the calibration information. Theassay calibration information can be compared with the display output todetermine the accuracy of the assay by including a pre-determined rangefor the display output in the information. Another method of determiningthe accuracy of the assay is to time the presence of the sample andcompare the time required to achieve the assay result to the calibrationinformation which can include a pre-determined range for that parameter.

Although one test card is analyzed by the embodiments illustrated above,the present invention also provides for sequentially analyzing multiplesampling areas on one test card or for analyzing more than two testcards either simultaneously or sequentially. Based upon the inventiveconcepts and embodiments described herein, it is within the scope ofthose skilled in the art to make the appropriate modifications.

The test card 16 of the present invention can have variousconfigurations. FIG. 3 illustrates one embodiment wherein the electrodepair 22 is provided in the detection area 20. one end 38 of each of theelectrode pair 22 electrically connects to a corresponding pair ofcontact pads 40 to provide connection to the processor 32 and the powersource 28 as previously described in FIG. 2. The connection between theelectrodes 22 and the contact pads 40 is made when the end 48 of thetest card 16 is inserted into the inlet port 14.

FIG. 4 illustrates another embodiment of the test 16 wherein a capillarychannel 50 is provided to transport a sample to the detection area 20.One end 52 of the capillary channel terminates in the proximity of theedge 54 of the test card which is not inserted into the inlet port 14.The opposite end 56 of the capillary channel connects to the detectionarea 20.

FIG. 5 shows another embodiment of a test card 16 which provides anon-board control test area 58 having a pair of electrodes 60 forcontacting the sample. One end 62 of each of the electrode pair 60electrically connects to a corresponding pair of contact pads 40 toprovide connection to the processor 32 and the power source 28 aspreviously described in FIG. 2. The capillary 50 bifurcates at end 56 totransport the sample to both the detection area 20 and the control testarea 58. Having a control test area 58 separate from the detection area20 allows the immobilization of different reagents in the control testarea than in the detection area.

FIG. 7 is a cross-sectional view of the test card 16 which uses anon-porous substrate 70 preferably made of a polymeric material. A topsheet 72, also made of a non-porous material, is placed over thesubstrate 70 defining the detection area 20. The electrode 22 is locatedin the detection area 20 and extends to the end 38.

The present invention also provides a reagent system for use incoagulation or lysis assays that rely on the generation of an electricalsignal (voltage, current, etc.) indicative of the blood clotting orlysis process. These reagent compositions are particularly useful inassay devices in which a liquid blood or plasma specimen is transportedby capillary action into the test card which contains the reagent and inwhich the assay is performed.

The reagent compositions have been developed primarily for use in bloodcoagulation or blood lysis assays which rely on changes in the viscosityand ultimately the reduction or increase in ionic mobility and ordiffusion characteristics of an added electroactroactive species of theblood or plasma sample as measured by the generation of an electricalsignal (current or voltage). Blood is an electrolyte and can thereforeconduct or carry an electric current. Electrolytic conductance is ameasure of the ability of a solution of electrolytes to carry anelectrical current by virtue of the mobility of its ions under theinfluence of a potential gradient. The ionic mobility and diffusionkinetics is a function of the solution viscosity, ionic size and chargeand the magnitude of the applied potential. When blood begins to clot orcoagulate, it thickens or causes its viscosity to increase. Thisincrease in viscosity inhibits or retards the ionic mobility. Theretardation of ionic mobility is directly proportional to a reduction inthe electrical current of the total solution. During the clottingprocess, the blood clot retracts and the fibrin monomers come togetherto form a clot. Addition of the electroactive chemical species to thereagent composition enhances the conductivity of unclotted blood bymodulating the measured signal. This enhancement leads to increasedsensitivity and reliability of the detection technique. As clottingoccurs, the fibrin clot retards the movement of ions and consequentlythe current reduces. Until such time when the clotting is complete,there may be a slight increase in the current of the clot due toaggregation of the electroactive species in spite of restricted ionicmobility and or diffusion. Such a current time profile is extremelyuseful in determining the onset of clotting as well as the endpoint ofthe clotting process and could conceptually provide a very accuratemeans of determining blood clotting times in PT, APTT and other clottingassays. The sensitivity of these type of current or voltage timemeasurements is inherent in the direct measurement technique and doesnot rely on secondary or indirect indicators such as color orfluorescence detection by optical means and turbidity measurements bylight scattering methods. Simultaneously the effect of thrombolytictherapy on blood clots, using for example Tissue Plasminogen Activator,the time taken to solubilize the clot (also referred to as the ClotLysis Time or Lysis Onset Time) can also be determined by this method.If it is assumed that the baseline current of the clot is high due topresence of aggregated electroactive species than as the clot dissolvesthere will be initially a reduction in current followed by a sharpincrease due to increased ionic mobility or diffusion characteristics.

For PT assays the reagent matrix normally consists of thromboplastinpurified from an aqueous extract of acetone dried brain tissue, whichcontains many components and impurities. In contrast, syntheticrecombinant thromboplastin (r-DNA thromboplasin) consists of arelatively well defined complex form by purified recombinant tissuefactor protein and a purified artificial lipid component. The presentinvention provides a reagent composition including either thromboplastinpurified and isolated from brain tissue or r-DNA thromboplastin capableof providing a PT result independent of any adverse factors.

The reagent compositions are preferably aqueous solution which can beapplied to the test card using various types of micro-dispensingtechniques which include, but is not limited to, ink jet, striper, andsprayer deposition methods, or dip coating and air dried in situ duringthe manufacturing process. In one such solution, thromboplastin reagentand electroactive speciess used in coagulation assays are mixed in ahomogeneous aqueous solution containing an appropriate proportions ofspontaneously hydratable and soluble carbohydrate chemical species suchas sucrose, starch, dextrose, dextran, maltodextrin, other water solublepolymers, binding agents and the like. The use of the carbohydratespecies is to facilitate hydration and stabilize the reagent matrixduring the solvation with the liquid sample such as blood or plasma.

The present invention also provides for detecting or measuring thechanges in the diffusion constant or kinetic profile of an electroactivespecies which is added to the blood sample as a function of time whilethe fluid undergoes clotting. Any electrochemical technique that allowsthe determination of the diffusion kinetics/constants of anelectroactive substance is suitable for use with the present invention.A known concentration of an electroactive species is dissolved in thesample and an apparent diffusion coefficient can then be measured. Theinformation obtained depends on the nature of the electroactive species.Suitable electrochemical techniques include polarography, cyclicvoltammetry (CV), rotating disk voltammetry (RDV),chronoamperometry/chronocoulometry, and chronopotentiometry and aredisclosed by E. Dayalan et al., "Micelle and Microemulsion DiffusionCoefficients", Electrochemistry in Colloids and Dispersion, VCHPublishers, Inc. New York 1992 and the references cited therein, whichis hereby incorporated in its entirely by reference. Thecurrent-diffusion coefficient relationships that are applicable for eachof these techniques are as follows:

Polarography (Ilkovic equation)

    i.sub.d =708nCD.sup.1/2 m.sup.2/3 t.sup.1/6

Cyclic voltammetry (Randles-Sevcik equation)

    i.sub.p =0.4463(n.sup.3/2 F.sup.3/2)/(R.sup.1/2 T.sup.1/2) ACD.sup.1/2 v.sup.1/2

Rotating disk voltammetry (Levich equation)

    i.sub.l =0.62nFACD.sup.2/3 v.sup.-1/6 W.sup.1/2

Chronoamperometry (Cottrell equation)

    i(t)=nFACD.sup.1/2 π.sup.-1/2 t.sup.-1/2

Chronopotentiometry (Sand equation)

    i(τ)=1/2nFACD.sup.1/2 π.sup.-1/2 τ.sup.-1/2

The symbols in the above mean the following: i_(d) is the polarographicdiffusion-limited current (A), n is the number of electrons transferred,C is the concentration of the electroactive probe (mol cm⁻³), D is thediffusion coefficient of the electroactive probe (cm² s⁻¹), m is themass flow rate of mercury at the dropping electrode (mg s⁻¹), t is thedrop time (s), i_(p) is the peak current (A), F is Faraday's constant(coulombs mol⁻¹), A is the area of the electrode (cm²), R is the gasconstant (J mol⁻¹), T is the temperature (K), v is voltage scan rate (Vs⁻¹), i_(l) is the limiting current (A), v is the kinematic viscosity ofthe solution (cm² s⁻¹), w is the angular velocity of the rotating diskelectrode (rad s⁻¹), i(t) is the diffusion current (A) at time t(s), andτ is the transition time (s).

Preferred electrochemical methods are voltammetry, cyclic voltammetry,chronoamperometry, and chronopotentiometry. In these methods the currentmeasured is proportional to the diffusion coefficient of theelectroactive species which is related to the viscosity of the samplemeasured over time. Generally, in chronoamperometry, the potential orvoltage is applied at a constant level, for example 400 millivolts oranother suitable voltage, which is sufficient to oxidize or reduce theelectroactive species which will result in a change of current overtime. In cyclic voltammetry the voltage is cycled at two differentpotentials. In chronopotentiometry the current is applied in a constantor prescribed change manner and the potential is measured over time.

A voltammetric method applies a potential and measures the current as afunction of time. Two variations of this method keeps the appliedpotential constant or changes the potential in a prescribed manner.Preferably, a voltammetric method is used to measure the diffusionlimited current of the electroactive species. The device measures thediffusion limited current of the electroactive species present in thereagent. The diffusion limited current is related to the diffusioncoefficient of the electroactive species in the sample which is directlya measurement of the viscosity change of the sample. FIG. 6 illustratesa disposable test card 16 which provides a miniaturized electrochemicalcell including a working electrode 64, a reference electrode 66 andpreferably a counter electrode 68 in the detection area 20. Theelectrodes can be made from different types of metal. Preferably, theworking electrode 64 and counter electrodes 68 are made ofgraphite/carbon, gold or platinum and the reference electrode 66 is madeof silver/silver chloride.

Preferred electroactive species are water soluble and form a redoxcouple. More specifically, ferricyanide, ferrocyanide, cadmium chloride,and methylviologen are most preferred electroactive species for use withthe present invention.

The reagent compositions immobilized on the inventive test cards havebeen developed primarily for use in blood coagulation or blood lysisassays which rely on changes in the viscosity and ultimately thereduction or increase in ionic mobility or changes in the diffusionkinetics of the blood plasma sample as measured by the generation of anelectrical signal (current or voltage). Since blood is an electrolyteand can therefore conduct or carry an electric current. Electrolyticconductance is a measure of the ability of a solution of electrolytes tocarry an electrical current by virtue of the mobility of its ions underthe influence of a potential gradient. The ionic mobility and diffusionkinetics is a function of the solution viscosity, ionic size and chargeand the magnitude of the applied potential. When blood begins to clot orcoagulate, it thickens or causes its viscosity to increase. Thisincrease in viscosity inhibits or retards the ionic mobility. Theretardation of ionic mobility is directly proportional to a reduction inthe electrical current of the total solution. During the clottingprocess, the blood clot retracts and the fibrin monomers come togetherto form a clot. As clotting occurs, the fibrin clot retards the movementof ions and consequently one can expect a reduction in the current. Suchcurrent time profile is extremely useful in determining the onset ofclotting as well as the endpoint of the clotting process and couldconceptually provide a very accurate means of determining blood clottingtimes in PT, APTT and other clotting assays. The sensitivity of thesetype of current or voltage time measurements is inherent in the directmeasurement technique and does not rely on secondary or indirectindicators. To enhance the stability of the reagents on the test card,it is preferred to add a preservative such as thimerosol, a surfactantsuch as triton and additional buffer salts.

The present invention can also be applied to a skin bleeding timeprocedure in which blood issuing from a standardized wound isprogressively quantitated by the increasing conductivity between asurface electrode and underlying skin. The test card described hereincan be placed directly on the wound of a patient. If necessary, theformation of a standardized wound can be made by incorporating anappropriately shaped lancet as part of the test card. The bloodemanating from the wound can be directly applied to the surface of thetest card.

Having generally described the present invention, a furtherunderstanding can be obtained by reference to the following specificexamples, which are provided herein for purposes of illustration onlyand are not intended to be limiting of the present invention.

EXAMPLES

Unless otherwise indicated, the following tests used a commerciallyavailable reagent thromboplastin-XS with calcium (Pt) which was obtainedfrom Dade International of Miami Fla. and sold under the tradenameINNOVIN® which is a dried recombinant human tissue factor with calcium.The Pt reagent accelerates the clot formation rate usually forming aclot in about 45 seconds. The Pt reagent contains a source of tissuethromboplastin and was reconstituted with deionized water as required bythe manufacturer, dispensed on a microelectrode, and partially airdryed. No additional additives like a preservative or surfactant wasused. The microelectrode was fabricated by Applied Graphics of Soquel,Calif. by printing a thin layer of silver to form the electrodes ontothe surface of a non-porous test card in the configuration illustratedin FIG. 3. The test card was washed with water and alcohol prior to use.All tests were carried out at ambient temperature. Once the blood samplewas applied to the microelectrode containing the Pt reagent, theclotting profile was manually recorded using a conventional conductivitymeter connected to the contact pads of the electrodes. The meter ismanufactured by Horiba in Japan.

FIG. 8 illustrates a change in conductivity with increasing viscosity asa whole blood sample coagulates. FIG. 9 plots the change in conductivityduring the test. The conductivity was measured immediately upon drawinguntil the sample began to coagulate. The conductivity starts at about0.31 mS/cm and, as discovered herein, characteristically decreasessomewhat uniformly until the conductivity levels off at about 0.24 mS/cmat which point the conductivity begins to rise. It was observed thatremoval of the clogging left only serum which is primarily electrolyteand affected the increase in conductivity.

Clotting Time is determined by plotting the conductivity decreasebetween t=0 and about t=150 seconds from these test results andsurprisingly yields an accurate Clotting Time measurement which can becorrelated to the PT Time. Although the tests were not optimized and didnot use a temperature control, the conductivity changes during theblood/plasma coagulation process are clearly demonstrated.

Other tests were run as a control with Pt reagent and salt water tocheck that the characteristic curve was the result of coagulation. Theresulting curves were flat which indicated that the response seen inFIG. 8 was indeed the result of coagulation. Over the same period oftime, the viscosity of tap water will not change and remains fairlyconstant.

FIG. 10 compares a normal plasma sample which has been citrated (CNP)with a heparinised normal plasma sample (HNP) and a citrated COUMADINplasma sample (CCP). The graph demonstrates that the CNP curve exhibitedthe fastest clotting. The CCP curve of a patient undergoinganticoagulant therapy with COUMADIN exhibits the characteristic slopeillustrated above, but clearly differs from the normal plasma sample.The HNP curve of a patient treated with Heparin exhibits a more extremeresponse to an anticoagulant drug.

FIG. 11 illustrates the clotting profiles of whole blood samplescomparing a Heparinized sample and a citrated sample. Again, thecharacteristic effect is present even in the whole blood of a patientbeing treated with Heparin. Several sets of these tests were run toconfirm reproducibility.

The present invention is useful in many types of coagulation and lysisassays. For example, and not for limitation, applications of the presentinvention include PT, APTT, thrombin time, fibrinogen assays, andplatelet detection. For fibrinolytis assays a lytic agent such as atissue plasminogen activator will be incorporated in the clottingreagent mixture. After clotting occurs there is an increase inconductivity indicating the onset of lysis.

Numerous modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

What is claimed is:
 1. A single-use electronic device for performing acoagulation or lysis assay of a blood sample, the device comprising:ahousing having an exterior surface and defining an interior area; meansfor receiving the blood sample through the housing into the interiorarea; a non-porous substrate positioned within the interior area forreceiving the blood sample thereon; a reagent for accelerating thecoagulation of the blood sample positioned on the substrate and incontact with the blood sample; an electroactive species positioned onthe substrate and in contact with the blood sample; means for measuringthe viscosity of the blood sample and generating an electrical signalwhich correlates to a curve of a coagulation or lysis assay; processingmeans being positioned within the interior area and connected to themeasuring means for receiving and converting the electrical signal intoa digital output corresponding to the coagulation or lysis assay usingassay calibration information stored in the processing means; anddisplay means for visually displaying the digital output external to thehousing, the display means being connected to the processing means. 2.The device of claim 1 wherein the measuring means includes:means forgenerating a signal of predetermined frequency, the generating meansbeing positioned within the interior area; a plurality of electrodesbeing positioned on the substrate and contacting the blood sample, theelectrodes connecting to the generating means for passing the signalinto the blood sample, the electrodes generating an electrical signalcorresponding to the conductivity/resistivity of the blood sample. 3.The device of claim 1 wherein the measuring means includes:means forgenerating a signal of predetermined frequency, the generating meansbeing positioned within the interior area; a plurality of electrodesbeing positioned on the substrate and contacting the blood sample, theelectrodes connecting to the generating means for passing the signalinto the blood sample, the electrodes generating an electrical signalcorresponding to the diffusion coefficient of the electroactive species.4. The device of claim 1 wherein the electroactive species is selectedfrom the group consisting of ferricyanide, ferrocyanide, cadmiumchloride, and methylviologen.
 5. The device of claim 1 wherein thedevice further includes a heater being positioned within the interiorarea and in thermal contact with the substrate so that the substrate canbe warmed to a pre-determined temperature upon receiving the bloodsample.
 6. The device of claim 1 wherein the processing means furthercalibrates the electrical signal to a reference standard using thestored assay calibration information.
 7. The device of claim 1 whereinthe processing means further receives the ambient temperature of theassay device from a sensor within the housing and adjusts assay resultsusing the stored assay calibration information.
 8. The device of claim 1wherein the assay calibration information stored in the processing meansdetermines results for at least one of assays selected from the groupconsisting of a Prothrombin Time, an Activated Partial ThromboplastinTime, platelet aggregation, and fibrinogen.
 9. A test card for anelectronic device which generates a signal having a pre-determinedfrequency and determines a curve of a coagulation or lysis assay of ablood sample, the test card comprising:a non-porous substrate defining asurface for receiving the blood sample; an electroactive speciespositioned on the substrate and in contact with the blood sample; areagent initially immobilized on the surface for contact with the bloodsample; and a plurality of electrodes being positioned on the substrateand contacting the blood sample, the electrodes adapted to receive andpass the signal into the blood sample, the electrodes generating anelectrical signal corresponding to the viscosity of the blood samplewhich correlates to the curve of the coagulation or lysis assay.
 10. Thetest card of claim 9 wherein the electrical signal generated by theelectrodes measures the conductivity/resistivity of the blood sample.11. The test card of claim 9 wherein the electrical signal generated bythe electrodes measures the diffusion coefficient of the electroactivespecies.
 12. The test card of claim 9 wherein the electroactive speciesis selected from the group consisting of ferricyanide, ferrocyanide,cadmium chloride, and methylviologen.
 13. A method of determining thecoagulation or lysis of a blood sample, the steps of the methodcomprising:introducing the blood sample to a sample receptor site on asubstrate and contacting an electroactive species positioned on thesubstrate with the blood sample; accelerating coagulation of the bloodsample by chemically reacting the blood sample with at least one reagenton the substrate to produce a detectable change in the viscosity of theblood sample which correlates with a state of coagulation or lysis ofthe blood sample; measuring the viscosity of the blood sample andgenerating an electrical signal which correlates to a curve of acoagulation or lysis assay; and processing the electrical signal into adigital output corresponding to the coagulation or lysis assay usingassay calibration information.
 14. The method of claim 13 wherein themethod includes the step of displaying the digital output.
 15. Themethod of claim 13 wherein the measuring step includes:generating asignal of predetermined frequency; contacting a plurality of electrodeswith the blood sample; passing the signal of predetermined frequencythrough the electrodes into the blood sample; and generating theelectrical signal with the electrodes corresponding to theconductivity/resistivity of the blood sample.
 16. The method of claim 13wherein the measuring step includes:generating a signal of predeterminedfrequency; contacting a plurality of electrodes with the blood sample;passing the signal of predetermined frequency through the electrodesinto the blood sample; and generating the electrical signal with theelectrodes corresponding to the diffusion coefficient of theelectroactive species.
 17. The method of claim 13 wherein the methodincludes calibrating the electrical signal to a reference standard usingthe assay calibration information to determine results for at least oneof assays selected from the group consisting of a Prothrombin Time, anActivated Partial Thromboplastin Time, platelet aggregation, andfibrinogen.